Separations-based analyses are a prominent part of biological research, allowing one to characterize different biological samples, reaction products and the like. Examples of some of the more prevalent separations-based analyses include electrophoretic separations of macromolecular species, e.g., proteins and nucleic acids. Electrophoresis, e.g., capillary electrophoresis, has been established as a highly effective method for separating macromolecular species in order that they might be further characterized. Protein and nucleic acid molecules are two major examples of molecular species that are routinely fractionated and characterized using electrophoretic systems.
Both microfluidic and macrofluidic devices have been applied in separations-based analyses. Examples of novel microfluidic devices and methods for use in the separation of molecular and macromolecular species by electrophoretic means are described in U.S. Pat. Nos. 5,958,694, 6,032,710, and 7,419,784, for example, the entire contents of which are incorporated by reference herein. In such devices, the sample containing the molecular or macromolecular species for which separation is desired is placed in one end of a separation channel located in a fluidic substrate, and a voltage gradient is applied along the length of the channel. As the sample components are electrophoretically transported along the length of the channel and, optionally, through a separation (sieving) matrix disposed therein, those components are resolved. The separated components are then detected at a detection point along the length of the channel, typically near the terminus of the separation channel downstream from the point at which the sample was introduced. Following detection, the separated components may be directed to a collection reservoir/well in the device (or to an external device such as a multiwell plate using a capillary pipettor, for example) for subsequent extraction or disposal.
In many situations, it is desirable to extract selected fragments of interest, such as DNA (deoxyribonucleic acid) fragments, following the separation of the fragments into bands in the separation matrix for further processing or analysis, e.g., restriction enzyme modification, T4 ligation, PCR (polymerase chain reaction) amplification, mass spectroscopy, or polynucleotide kinase reactions. The typical process used by laboratory researchers for extracting and isolating selected DNA fragments of interest (and other desired nucleic acid and protein fragments) from a separation matrix (such as an agarose gel) involves staining the separated fragments and then shining UV (ultraviolet) light on the fragments to visualize the separated bands. A razor blade is then used to manually cut the gel above and below each fragment of interest. The DNA must then be extracted and purified from the gel slice. The recovered DNA can then be used for further processing or analysis. This extraction process, however, is time consuming, laborious, and potentially damaging to the DNA (e.g., nicking of the DNA can occur if the DNA is exposed to UV light too long while the fragments of interest are being illuminated for excision).
Thus, in performing separations-based analyses, it would be desirable to be able to also isolate or extract one or more of the separated components in the device itself for further analysis or processing. The recovered or isolated fragments could then be used for a variety of different processes including, for example, the following: amplification using polymerase chain reaction (PCR); ligation reactions for cloning small to medium-sized strands of DNA into bacterial plasmids, bacteriophages, and small animal viruses to allow the production of pure DNA in sufficient quantities to allow its chemical analysis; reactions to dissolve a separated protein or nucleic acid component in a suitable matrix for further analysis by a mass spectrometer using, for example, Matrix-Assisted Laser Desorption Ionization (MALDI); binding reactions to bind a labeling agent to one or more separated protein or nucleic acid components for further analysis; or other similar post-detection processes. In addition, in the case of PCR samples, it is important to be able to separate smaller dimer and primer molecules from the main nucleic acid fragments in the sample and then isolate and collect the main nucleic acid fragments for further analysis or processing, while directing the smaller primer and dimer components to a waste reservoir/well for removal and subsequent disposal.
Typically, the electrodes used in performing electrophoretic separations in a fluidic device are included in an instrument that receives the fluidic device, rather than in the fluidic device itself. Because the instrument electrodes are not disposable and are used serially with multiple devices, contamination of the samples, reaction mixtures, and reaction products may result. In some separations-based applications, limited contamination is not a problem; however, when a component of interest is to be isolated or extracted from a sample, avoiding contamination of a subsequent sample with components of an earlier sample can be crucial. Therefore, it would be advantageous to provide improved fluidic devices that incorporate electrodes into the devices, eliminating the need for multi-use electrodes being included in the instrument that receives the devices.